This proposal focuses on the role of the Galphas/adenylyl cyclase pathway and how constitutive and conditional elevation of cAMP affects cell proliferation and transformation. We have found that mutant (Q227L) activated Gas (alphas ) extensively inhibited H-Ras induced transformation of NIH-3T3 fibroblasts. We studied the effects of alphas expression on human breast cancer cell lines, since transformation of mammary epithelial cells is often associated with increases in receptor-tyrosine kinases that transmit their signals through Ras to MAP-kinase 1,2. Expression of alphas or elevation of cAMP results in a decrease in the intrinsic MAP-kinase (ERK1,2) activity of MCF-7 cell line and increased expression of the cell cycle inhibitor p27kip in MDA-231 and MDA-435 cell lines. This accompanied by inhibition of colony formation in soft agar and tumor formation in Nu/Nu mice. We constructed recombinant adenovirus that contains a FLAG epitope tagged alphas (ADV-alphas ). Infection of several types of human breast cancer cells representative of the latter stages of the disease, with the ADV-alphas in vitro, results in an inhibition of ability to form tumors in Nu/Nu mice. We find that application of ADV-alphas into established tumors results in blockade of their growth. These observations indicate that interactions between signaling pathways may regulate mammary carcinogenesis. We will expand these studies to include several human cell lines representing the later stages of breast cancer. We will test if adenovirus direction expression of alphas in combination with low doses of taxol causes full regression of tumors. We will determine the molecular mechanisms by which expression of alphas inhibits expression of the transformed phenotype in human mammary epithelial cells. We will look for inhibition of signal flow through the Raf-MAP kinase pathway as well as alterations in the expression of the cell cycle regulators. We will study the molecular consequences of expressing adenylyl cyclase 2 which can be stimulated by protein kinase C, in mammary epithelial cells and determine if such expression blocks proliferative signals conditionally and suppresses tumorigenesis. We will develop transgenic mice that express alphas or adenylyl cyclase 2 in mammary tissue to determine if this expression lowers the incidence of mammary tumors caused by expression of oncogenes such as erbB2 (HER2). From these studies we hope to gain an understanding of the mechanisms involved in the interactions between signaling pathways in regulating the proliferation and transformation of mammary epithelia. We also hope to determine whether such interactions can be used in the treatment of breast cancer and the feasibility of expressing components of the Galphas pathways as "germ-line therapy" for prevention of breast cancers in high-risk populations.